The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
The polymerase chain reaction (PCR; Saiki et al., 1985) is a good example of a chemical reaction that requires careful temperature control: in PCR, a specific DNA sequence is amplified by subjecting the DNA sample to cyclic temperature changes. First, the double stranded DNA template is denatured by increasing the temperature of the reaction mixture up to approximately 95° C. Then, the temperature is lowered to approximately 40-70° C. At this temperature, short synthetic oligonucleotide primers hybridize to their complementary sequences rendered into a single stranded state in the previous heating step. After this, the temperature can be increased to approximately 72° C. At this temperature, a heat stable DNA polymerase extends the primers, thus creating a complementary copy of the original single stranded template DNA. (In many applications, this extension step can be performed at the same temperature as the hybridization step. Quite often, both primer hybridization and primer extension are performed at approximately 60° C.). By repeating the temperature cycle many times, the amount of template DNA is, if the amplification efficiency is ideal, doubled at each cycle. In addition to PCR, many if not all biological and chemical reactions require a certain temperature to occur in a predictable manner. Examples of such reactions with defined temperature requirements include other nucleic acid amplification reactions [such as nucleic acid sequence based amplification (NASBA) (Compton, 1991), ligase chain reaction (LCR) (Barany, 1991), strand displacement amplification (SDA) (Walker et al., 1992) and rolling circle amplification (RCA) (Banér et al. 1998)], immunocomplex formation (i.e. binding of an antibody to an antigen) (Price and Newman, 1997) and nearly all other enzymatic and chemical reactions.
A number of solutions exist for controlling a reaction temperature. In PCR, one normally places the reaction vessels in a block of metal, the temperature of which is changed periodically. However, a major drawback of this approach is that a significant amount of time is required for the block to change its temperature: once the target temperatures have been reached, the reaction itself occurs very fast. Therefore, it is the thermal mass of the block that is limiting the speed of the reaction rather than the reaction itself.
The rate of temperature change inside a reaction vessel can be increased by a technique known to those skilled in the art as over or under shooting: To cool the contents of a reaction vessel to a low target temperature, the metal block is first cooled to a temperature below the target temperature, after which the metal block is heated to the target temperature. Alternatively, to heat the contents of the reaction vessel to a high target temperature, the metal block is first heated to a temperature that is above the target temperature, after which the block is cooled to the high target temperature. In this manner, the rate of temperature change inside a reaction vessel can be increased. However, the process is still rather slow due to the fact that it takes time for the metal block to change its temperature.
Another approach for thermal cycling includes the use of hot and cold air to change the temperature of a reaction mixture that is placed inside a glass capillary with a great surface-to-volume ratio (Wittwer et al., 1997). This allows very rapid temperature change. However, when glass capillaries are used, the maximum reaction volume is often so small that it starts to limit the analytical sensitivity of the application. Also, the chemical properties of glass may inhibit some chemical reactions. The fragility of glass capillaries is also a problem as they break very easily when handled. Another approach is based on physically moving the reaction mixture through a channel that passes through areas of different temperatures. Such a technique has been described by for example Kopp et al. (1998). In these applications, however, the reaction volumes are even smaller and thus analytical sensitivity is heavily compromised. Also, one either has to reuse the same amplification channel for many samples, which introduces a serious risk of carry-over contamination between different samples or alternatively, the amplification vessels have to be of a disposable, single-use design, which increases the total costs of the assay significantly since the production costs of microfluidic channels can be much higher than the production costs of simple plastic reaction tubes, depending on manufacturing volume of course. Yet another approach is based on having several metal blocks or water baths at defined temperatures and changing the place of the reaction vessel cyclically between the blocks or baths of different temperatures. Examples of commercially available thermal cyclers based on this principle include the RoboCycler (Stratagene, USA) and the H2OBIT Thermal Cycler (ABgene, United Kingdom). In these applications one achieves a higher rate of temperature change than with a single block. However, since sample tubes with small surface-to-volume ratios are used, the rate of temperature change is still not as fast as it can be.
Techniques in which a reaction vessel with a high surface-to-volume ratio is transferred cyclically between thermal blocks at defined temperatures have been described in U.S. Pat. No. 4,902,624, EP 0 31 8255 and in U.S. Pat. No. 5,736,106. In these approaches, certain problems are encountered. First of all, the rate of temperature change is not ideal for all applications. Secondly, when the reaction mixture is heated up to high temperatures, the pressure inside the reaction vessel increases, which may result in that the reaction vessel breaks and the reaction mixture evaporates.
Where cyclic changes of temperature are not needed, for example in most immunoassays, it is common practice to place the reaction vessel in the atmosphere of an incubator set at a defined temperature. In such procedures, a significant amount of time is spent on heating up or cooling the contents of the reaction vessel since, due to the poor surface-to-volume ratio of the vessels and the poor thermal conductivity of static air, the rate of thermal exchange between the vessel and its surroundings are not ideal.